Direct measurement of DNA molecular length in solution using optical tweezers: detection of looping due to binding protein interactions.
DNA looping is caused by the interaction between DNA binding proteins located at separate positions on a DNA molecule and may play an important role in transcription regulation. We have developed a system to stretch single DNA molecules and to measure changes in molecular length. DNA molecules were prepared and 5' end-labeled by PCR amplification. Two beads and the intervening DNA molecule were trapped and manipulated independently with dual trap optical tweezers. The trapped DNA molecule was then stretched and the extension (the distance between the two beads) was measured. The extension at the specific tension force of 30 pN was calculated and used as a molecular length. The molecular length was found to be proportional to the base pair number. The rise per residue was calculated to be 3.31 +/- 0.05 A. The length measurement was applied to DNA fragments containing GC box sequences at two different locations separated by a distance of 2.428 kbp. The addition of GC box binding transcription factor Sp1 shortened the molecular length, suggesting DNA looping forms as a result of interaction between transcription factors.
Sakata-Sogawa
K
K
Laboratory for Photo-Biology, Photodynamics Research Center, Institute of Physical and Chemical Research (RIKEN), Sendai, Japan. ksogawa@postman.riken.go.jp
Kurachi
M
M
Sogawa
K
K
Fujii-Kuriyama
Y
Y
Tashiro
H
H
D001483:Base Sequence; D003596:Cytosine / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D006147:Guanine / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D055095:Optics and Photonics; D012996:Solutions; D016329:Sp1 Transcription Factor / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D012996:Solutions; D016329:Sp1 Transcription Factor; D006147:Guanine; D003596:Cytosine; D004247:DNA